This project presents an integrated genetic and biochemical attack on identifying and mapping mitochondrial structural genes and associated regulatory regions in Saccharomyces cerevisiae. First, a physical map of selected wild-type mitochondrial genomes will be established using restriction endonucleases. Segments of the mitochondrial genome are purified using genetically defined petite mutants whose regions of retained wild-type sequence will be accessed by restriction analysis. A process for determining structural genes retained by petites (termed "gene rescue") will be used to assign structural genes to specific restriction fragments. This procedure involves mating petites to mit-(or wild-type) strains lacking (or having an altered form of) specific products of mitochondrial protein synthesis. Zygotes are labeled with radioactive precursors shortly after mating and the products of mitochondrial protein synthesis are analyzed by autoradiography of slab gradient gels. It has been shown for several proteins that petites can retain complete structural genes and that they can be expressed in zygotes. Analysis of petites with varying lengths of retained information will permit the precise mapping of genes and their regulatory regions. The identification of a new genetic locus (varl) mapping between the ery&olil loci on the wild-type map was instrumental in the development of the gene rescue protocol. This locus exists in several allelic forms in wild-type strains and specifies a soluble protein of molecular weight ranging from 38,000 to 48,000. Taken as a whole, it will be possible to assign specific protein gene products to known genetic loci and to establish the detailed physical map of mitochondrial DNA. Since petites may retain as few as one structural gene, these at least should lead to the identification of mitochondrial genes and gene products other than those already characterized (but not yet physically mapped).